Our laboratory has embarked on a new initiative to discover oncogenic somatic mutations in lymphoid malignancies by cancer gene resequencing. Previously, we discovered that a pathway involving CARD11, BCL10 and MALT1 (the CBM complex) was responsible for the constitutive NF-kB signaling in ABC DLBCL. We then identified recurrent somatic mutations in the CARD11 gene in 10% of ABC DLBCL biopsies that constitutively engaged NF-kB signaling. Subsequently, we defined a chronic active form of B cell receptor (BCR) signaling that activates NF-kB in ABC DLBCLs with wild-type CARD11. Such ABC DLBCLs die upon knockdown of BCR signaling components, including subunits of the B cell receptor itself. ABC DLBCLs have prominent clusters of the BCR in the plasma membrane, similar to antigen-stimulated normal B cells. Cancer gene resequencing revealed that over one fifth of ABC DLBCLs have mutations in the CD79B or CD79A subunits of the BCR that affect their critical ITAM signaling motifs, generating BCRs that avoid negative autoregulation by the LYN tyrosine kinase. Recently we discovered an important role for the linear ubiquitin chain assembly complex(LUBAC) in the survival of ABC DLBCL. This ubiquitin ligase associates withe CBM complex and is required for NF-kB engagement. We identified a rare germ line polymorphism in the coding region of the LUBAC subunit RNF31 that is enriched in ABC DLBCL. This creates a mutant isoform that promotes LUBAC assembly and NF-kB activation. We designed a peptide inhibitor based on this mutant that specifically kills ABC DLBCL cells, suggesting that LUBAC is a promising therapeutic target in this lymphoma subtype. We also identified oncogenic signaling by the adapter protein MYD88 as the genetic basis for the JAK-STAT3 activation in ABC DLBCL. ABC DLBCLs depend on MYD88 and its associated kinases IRAK1 and IRAK4. By resequencing we identified MYD88 mutations in 39% of ABC DLBCLs, with 29% changing one leucine in the MYD88 TIR domain to proline (L265P). The L265P mutant isoform spontaneously coordinates a signaling complexed involving IRAK1 and IRAK4, which turns on the NF-kB, JAK-STAT3 pathway, and type I interferon pathway. Small molecule inhibitors of IRAK4 kinase are selectively lethal to ABC DLBCL cells, offering new therapeutic prospects. There are several new drugs entering early phase clinical trials that target the pathways we have implicated using our functional genomics methods. The B cell receptor signaling pathway affords many possible targets for the treatment of ABC DLBCL, notably BTK. We conducted a phase Ib clinical trials of a small molecule BTK inhibitor, termed ibrutinib, in patients with relapsed/refractory DLBCL, assigned to a molecular subtype by gene expression profiling. Ibrutinib monotherapy induced several complete and partial responses in patients with ABC DLBCL, including those with primary refractory tumors that had never responded to any prior therapy. One patient has been in a sustained complete response for over 3 years, taking ibrutinib daily with no discernible side effects. Of note, ABC DLBCL tumors with and without CD79B mutations have responded, suggesting that BCR pathway addiction may be a prevalent feature in this lymphoma subtype. Lenalidomide has had activity in early phase clinical trials against ABC DLBCL, prompting us to investigate its mode of action in this setting. We discovered that lenalidomide induces the secretion of interferon beta by the ABC DLBCL cells, which is an important component of lenalidomide-induced cell death in ABC DLBCL. In addition, lenalidomide blocked B cell receptor signaling to NF-kB by decreasing expression of CARD11. Both of these phenotypes could be traced to the ability of lenalidomide to decrease expression of IRF4, a transcription factor that plays an essential survival role in ABC DLBCL. IRF4 is itself an NF-kB target gene, so that that agents that inhibit BCR signaling, such as ibrutinib, also decrease IRF4 expression.Combined treatment with lenalidomide and ibrutinib virtually eliminated IRF4 expression, leading to synergistic killing of ABC DLBCLs. We have developed new methods to identify drug synergies in cancer together with Craig Thomas and colleagues in NCATS. Using a combinatorial high-throughput drug screening platform, we can evaluate the effects of two drugs in combination, each tested at 10 different concentrations, thereby identify concentration ranges in which the drugs synergize in killing cancer cells. As a test case, we used this platform to demonstrate that ibrutinib synergizes with inhibitors of the PI(3) kinase pathway, BCL2 inhibitors, and standard cytotoxic chemotherapeutic agents. Using this platform, we observed that a small molecule inhibitor of BET-domain chromatin proteins, JQ-1, synergized with ibrutinib and other inhibitors of BCR signaling in killing ABC DLBCL cells. This unexpected finding was complemented by functional studies showing that JQ-1, which acts in the nucleus, potently inhibits IkB kinase, the key regulator of the NF-kB pathway, which resides in the cytoplasm. This suggests that constitutive NF-kB activity in ABC DLBCL may be mediated, in part, by signals emanating in the nucleus. BET protein inhibitors emerge as new drug class that should be explored for the treatment of ABC DLBCL. Burkitt lymphoma (BL) can often be cured by intensive chemotherapy, but the toxicity of such therapy precludes its use in the elderly and in patients with endemic BL in developing countries, necessitating new strategies. The normal germinal center B cell is the presumed cell of origin for both BL and diffuse large B cell lymphoma (DLBCL), yet gene expression analysis suggests that these malignancies may utilize different oncogenic pathways. In 70% of sporadic BL cases, mutations affecting the transcription factor TCF3 (E2A) or its negative regulator ID3 fostered TCF3 dependency. TCF3 activated the pro-survival PI(3) kinase pathway in BL, in part by augmenting tonic B cell receptor signaling. In 38% of cases, oncogenic CCND3 mutations produced highly stable cyclin D3 isoforms that drive cell cycle progression. These findings suggest opportunities to improve therapy for patients with BL. In particular, current BL therapy entails high dose chemotherapy that cannot be delivered safely to elderly patients or to children in Africa with the endemic form of BL. Potentially, combinations of targeted agents can be developed based on the molecular pathways that we have defined in BL that can be used safely and effectively in these settings. Based on our laboratory investigations, we have initiated a number of clinical trials using targeted agents that inhibit key oncogenic pathways in lymphoma. We have completed a multicenter phase 2 trial of ibrutinib in patients with relapsed/refractory DLBCL. Patients were assigned to the ABC or GCB subtypes by gene expression profiling and were given ibrutinib monotherapy. Ibrutinib produced a 37% response rate (complete + partial) in patients with relapsed/refractory ABC DLBCL, but only a 5% response rate in GCB DLBCL, as predicted by our laboratory investigations. Based on these promising results, ibrutinib is now being evaluated in newly diagnosed ABC DLBCL in combination with R-CHOP chemotherapy in a phase 3 randomized trial. While 10% of patients with ABC DLBCL survived on ibrutinib for 3 years or more, most had shorter responses, leading to us to search for other agents that might synergize with ibrutinib in killing these lymphoma cells. Based on laboratory evidence that ibrutinib synergizes with lenalidomide in killing ABC DLBCL cells, we have initiated a clinical trial of this combination together with chemotherapy.